In general, a two-signal mechanism is required to fully activate the T-cell. Signal-1 occurs when the T-cell antigen receptor (“TCR”) recognizes the peptide:MHC-II complex on the surface of an antigen presenting cell (“APC”). This first signal passes through the T-cell receptor and initiates a cascade of tyrosine phosphorylation/dephosphorylation events mediated by kinases and phosphatases and leads to the activation of calcium flux, nuclear factor of activated T-cells (“NF-AT”) and NF-κB transcription factors. These factors enter the nucleus of the T-cell and bind to promoters of genes responsible for effector functions. Signal-2 arises from the binding of Signal-2 receptors on the surface of T-cells to their ligands on the surface of an APC. Signal-2 receptors include CD28 and its ligand B7 as well as LFA-1 (CD11a/CD18) and its ligand ICAM-1. When a Signal-2 receptor and its ligand form a complex at the interface between the T-cell and APC receptor membranes, a series of signaling events occur. These events include serine/threonine phosphorylation/dephosphorylation and activation of guanine nucleotide exchange factors that activate adapter proteins with GTPase activity.
A defining stage of the immune response is the differentiation of CD4+ T-cells into either type-1 helper T-cells (TH1 cells) or type-2 helper T-cells (TH2 cells) as a result of the two signals. These two subtypes of TH cells and the regulatory network of cells that they selectively activate are well known to correlate with human health conditions and disease states. Differentiation into TH1 cells results in predominantly cell-mediated immunity while differentiation into TH2 cells results in predominantly humoral immunity. Each of these immunity types helps to protect the body against different types of invasion. Type-1 immunity protects the body against intracellular pathogens such as bacteria, but is also implicated in organ-specific autoimmune diseases. Type-2 immunity is important for protection against extracellular parasites, but is associated with allergic reactions as well. Development of TH1 cells is driven by a cytokine called interleukin-12, which is produced by immune cells known as macrophages and dendritic cells. Interleukin-12 induces or stimulates the naive T-cell (CD4+ T-cells) to produce interferon-gamma (“IFN-gamma”) and interleukin-2 (“IL-2”). These two cytokines (IL-2 and IFN-gamma) are involved in classic cell-mediated functions such as clonal expansion of cytotoxic T-lymphocytes (“CTLs”), macrophage activation, and class switching to IgG isotypes that mediate complement lysis of sensitized cells. Commitment to a TH1 immune response is enhanced by the presence of IFN-gamma which up-regulates expression of the interleukin-12 (“IL-12”) receptor while inhibiting the development of TH2 cells. TH2 immunity results from the production of interleukin-4 (“IL-4”) by the naive T-cell. IL-4 induces TH2 development and the subsequent production of IL-4, interleukin-5 (“IL-5”), interleukin-10 (“IL-10”), and interleukin-13 (“IL-13”). IL-4 also operates to down-regulate expression of the IL-12 receptor on developing cells, thereby inhibiting TH1 development and helping undifferentiated T-cells to commit to TH2 cell development. Additionally, IL-4 and IL-5 are known to activate B-cells and switch to neutralizing antibody (IgG1 in the mouse) and IgE, the initiator of immediate hypersensitivity.
Multiple sclerosis (“MS”) is the disease of the central nervous systems, including brain, spinal cord, and the optic nerves because of the damage in myelin. Myelin is a fatty tissue surrounding the nerve fibers and it helps the nerve conduct the electric impulses. The lost of myelin in many nerve areas is marked by nerve damages in a form of lesions or plaques in the nervous systems called sclerosis. In some cases, the nerve fiber can also be broken. Therefore, the nerve cannot conduct the electrical impulses that are needed for the nervous system to function. As a result the MS patient shows the various symptoms of the disease, including weakness, abnormal sensation, vision changes, and clumsiness. These can be detected by abnormal responses of the pupils, weakness in arms and legs, altered reflex responses, impaired coordination, and changes in speech patterns. The damage to the nerve is due to the attack by the immune systems.
Although the cause of MS is still not clear, scientists agree that MS is one type of autoimmune diseases that is marked by inflammation and destruction of myelin. It has been found that the immune cells of MS patients have been altered. The function of suppressor T-cell decreases in the peripheral blood during acute exacerbation followed by an increase in the number of activated helper T-cells in MS patients. The number of activated T-cells that cross the blood-brain barriers into the brain is increased. These T-cells are also found in the lesion region in of the nervous systems of MS patients.
To diagnose MS is very difficult because there is no single test available to rule out and identify whether a patient has MS or not. Currently, a combination of tests is used to diagnose patients with MS, including: (a) imaging of the brain using magnetic resonance imaging (“MRI”), (b) performing Evoke potential test, and (c) evaluating materials (i.e., antibodies and proteins) found in the spinal tap fluid.
MRI provides a detailed view of the change in the brain of MS patients; this imaging tool can visualize and count the damages in the white matter in a form of lesions or plaques in brain and spinal cord. One of the indications of MS is that there are two separate demyelinating lesions, suggesting damages in the nervous system within the brain, spinal cord, and optic nerves. The observance of abnormality by MRI does not necessarily mean a development of MS because there are other diseases that can cause lesions that look similar to MS. A similar type of spots called unidentified bright spots (“UBOs”) can also be found in older and healthy individuals. Thus, MRI result only cannot be used to determine occurrence of MS. On the other hand, 5% of MS patients do not show any lesions in the brain by MRI; the lesions either cannot be detected by MRI or may be found in the spinal cord. Thus, there is a need to develop a method that can specifically differentiate the presence of MS related lesions from UBOs.
The Evoke potential tests are to measure the quick and accurate the nervous system of the patient responds to a particular type of stimulation; these tests indicate the slowing down of the nerve impulse due to the destruction of myelin. Evoke potential (“EP”) tests can detect the slowdown of messages carried by the nerves in various part of the brain and provides evidence of the undetected lesions by MRI. Visual Evoke Potential (“VEP”) is the most common and acceptable method to diagnose MS patients.
The spinal tap is evaluated for antibodies and other marker proteins due the activation immune cells such as T-cells and B-cells. The antigen-specific activation T-cells by presentation myelin protein fragments by MHC-II molecules on the surface of APCs causes the production of antibodies and other immune-related proteins in the cerebrospinal fluids. This fluid can be sampled by a lumbar puncture or spinal tap. The presence of certain antibodies called oligoclonal bands from the spinal fluids indicates the presence of the disease and 90-95% of MS patients have oligoclonal bands. Unfortunately, these oligoclonal bands are also presence in other autoimmune diseases; thus, this test is not the only positive proof for the disease.
Because the attack of myelin is due to the activation of a subpopulation T-cells that recognize a specific antigen from proteins in myelin. The activation of antigenic-specific T-cells is due to recognition of antigens from myelin protein on the surface of APCs (i.e., B-cells, dendritic cells, and macrophages) via antigen:MHC complex. The immunological synapse is formed at the interface of T-cell:APCs via a combination of Signal-1 (TCR:MHC-peptide complexes) and Signal-2 (ICAM-1/LFA-1 complexes). Initially, the TCR:MHC-peptide complexes (Signal-1) are formed at an outer region or ring, and the ICAM-1/LFA-1 complexes (Signal-2) are formed at the inner region of the synapse. As the T-cell activation process proceeds, the ICAM-1/LFA-1 clusters migrate to the outer ring and the TCR/MHC-peptide complex moves to the inner ring. In the final state, TCR/MHC-peptide complexes congregate at the center to form a central supramolecular activation cluster (“cSMAC”), and the ICAM-1/LFA-1 complexes form a ring around the central zone to from a peripheral supramolecular activation cluster (“pSMAC”).
A major goal of modern applied immunology is to be able to switch from TH1 dominant immunity to TH2 responses. This is especially true in autoimmune diseases like multiple sclerosis (“MS”) and transplant rejection. Accordingly, what is needed in the art is modifiers of these immune responses so that type-1 immunity can be replaced with type-2 immunity as desired in order to combat different human disease states or health conditions.
In the past several years, the present inventors have conducted research in the area of bifunctional inhibitors (“BPIs”). In general, a BPI is derived from two peptides and includes a portion of a Signal-1 moiety (derived from a TCR epitope, i.e. a small peptide antigen) at one end and a portion of a Signal-2 moiety (derived from a Signal-2 receptor on the T-cell) at the other end. These two ends are directly connected to each other or connected via a non-substrate linker. The general concept of a BPI is set forth in Murray et al., U.S. Published Patent Application No. 2005/0107585, which is incorporated by reference. The work in that patent dealt largely with a BPI comprised of a GAD65 (208-217) Signal-1 moiety associated with type-1 diabetes linked to a LFA-1 alpha subunit CD11a (237-247) Signal-2 moiety.
The present invention is directed to novel BPIs and methods of use pertaining to use of the BPIs in modulating the immune response in MS and its animal model of experimental autoimmune encephalomyelitis (“EAE”), as well as a treatment and diagnostic method for MS and EAE. Moreover, the novel BPIs were capable of modulating the immune response towards a TH2 response.